Inhibitors of dna pk and uses thereof

ABSTRACT

The present disclosure relates to compositions and methods for improving the transplantation outcome and/or reducing immune response in a subject. The compositions comprise a DNA-PK inhibitor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/479,945, filed Mar. 31, 2017, which is hereby incorporated byreference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under grant P20 GM121293awarded by National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods for improvingthe transplantation outcome and/or reducing immune response in asubject.

BACKGROUND OF THE INVENTION

DNA-dependent protein kinase (DNA-PK) is a 460 kDa polypeptide member ofthe PI3k family. DNA-PK is believed to serve as a recruiting andscaffolding protein for DNA ligase.

IL-2 is a T cell-derived cytokine that influences a multitude of keyelements in the immune response including the proliferation anddifferentiation of B and T lymphocytes. Expression of IL-2 is initiatedupon calcineurin activation. Calcineurin is a calcium andcalmodulin-dependent protein serine/threonine phosphatase that uponactivation, dephosphorylates Nuclear Factor of Activated T-cells (NFAT)allowing it to translocate to the nucleus and upregulate expression oftarget genes (including IL-2). IL-2 then binds to its receptor IL-2R,expressed on the surface of lymphocytes, to induce signaling thatimpacts both arms of the immune response, humoral and cellular immunity.

Allograft rejection is an immune response, involving activatedT-lymphocytes. Currently used immunosuppressive protocols designed toinhibit rejection involve the administration of drugs such asazathioprine, cyclosporine, and corticosteroids, all of which causetoxic side-effects to non-lymphoid tissues. T-lymphocytes orchestrateboth the initiation and propagation of immune responses largely throughthe secretion of interleukin-2 (IL-2). IL-2 is primarily involved in theregulation of T-lymphocyte proliferation but also activates naturalkiller (NK), B- and lymphokine-activated killer (LAK) cells.Inappropriate responses of T-lymphocytes are associated with a range ofimmune diseases, including allergies and autoimmune diseases.

Therefore, what is needed, is a method to reduce or suppress IL-2production to reduce the immune response.

SUMMARY OF THE INVENTION

One aspect of the present disclosure is directed to a method ofimproving the transplantation outcome in a subject receiving an organtransplant. The method comprises administering to the subject atherapeutically effective amount of a composition comprising a DNA-PKinhibitor, wherein the DNA-PK inhibitor improves the transplantationoutcome.

Another aspect of the present disclosure is directed to a method ofreducing immune response in a subject in need thereof. The methodcomprises administering to the subject a therapeutically effectiveamount of a composition comprising a DNA-PK inhibitor, wherein theDNA-PK inhibitor reduces an immune response.

An additional aspect of the present disclosure is directed to a methodof reducing an immune response in a subject receiving an organ or tissuetransplant. The method comprises administering to the subject atherapeutically effective amount of a composition comprising a DNA-PKinhibitor, wherein the DNA-PK inhibitor improves the transplantationoutcome.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, and FIG. 1E depict inhibition ofDNA-PKcs in T cells and PBMCs blocks IL-2 production. (FIG. 1A) Jurkatcells were treated with the DNA-PKcs inhibitor NU7441 at varyingconcentrations for 48 hours and no significant reduction in viabilitywas detected. (FIG. 1B) Jurkat cells were stimulated with PMA (50ng/mL)+PHA (1 μg/mL), treated with NU7441, and analyzed for IL-2production 24 hours later. NU7441 treatment significantly blocked IL-2secretion. (FIG. 1C) IL-2 production stimulated by activation of Jurkatcells with anti-CD28/CD3 dynabeads at a 1:1 ratio for 24 hours wasinhibited by NU7441 treatment. (FIG. 1D) Treatment of Jurkat cells withshRNA reduced DNA-PKcs expression at 2.5 and 5 μg as seen by westernblot analysis. Loss of DNA-PKcs expression significantly reduced IL-2production. (FIG. 1E) NU7441 significantly reduce IL-2 productionfollowing activation with PHA+PMA in PBMCs. ** p<0.002 *** p<0.001 errorbars=s.d.

FIG. 2A and FIG. 2B depict inhibition of DNA-PKcs blocks translocationof NFAT to the nucleus. (FIG. 2A) Western blot analysis of Jurkat celllysates showed activation of T cells with PMA+PHA inducedphosphorylation of DNA-PKcs at site s2056 (pDNA-PK) and dephosphorylatedNFAT at s237 (pNFAT). Treatment with NU7441 inhibited thedephosphorylation of NFAT at site s237 which is critical for itstranslocation to the nucleus. GAPDH was used as a loading control. (FIG.2B) Immunocytochemistry analysis of Jurkat cells treated with NU7441.The inhibitor (2.5 μM) blocked translocation of NFAT to the nucleusfollowing activation with PMA+PHA. Nuclei were stained with Dapi. 40×images are shown.

FIG. 3A, FIG. 3B, and FIG. 3C depict DNA-PKcs inhibition blockscalcineurin activity in T cells. (FIG. 3A) Jurkat cells were activatedwith PMA+PHA, treated with the DNA-PKcs inhibitor NU7441 (2.5 μM) andmonitored for calcineurin phosphatase activity. Inhibition caused asignificant reduction in calcineurin activity. (FIG. 3B) Level of Ca²⁺in Jurkat cell lysates following activation with PMA+PHA was monitored.Ca²⁺ levels were not affected by the addition of the NU7441 inhibitor.(FIG. 3C) Western blot and Elisa analysis of active phosphorylated mTORin activated Jurkat cells indicated that inhibition of DNA-PKcs does notalter mTOR activation. ***p<0.001 error bars=s.d.

FIG. 4A and FIG. 4B depict inhibition of DNA-PKcs reducesphosphorylation of CHK2 and stabilizes the calcineurin inhibitor,Cabin1. (FIG. 4A) Western blot analysis of Jurkat lysates followingactivation with PMA+PHA and NU7441 treatment. Activation increasedphosphorylation of DNA-PKcs and CHK2. DNA-PKcs inhibition reduced CHK2phosphorylation and elevated Cabin1 expression. GAPDH was used as aloading control. (FIG. 4B) Schematic depicting the signaling pathway inT cells used by DNA-PKcs to regulate IL-2 production. DNA-PKcsphosphorylates CHK2 which in turns phosphorylates Cabin1 targeting itfor destruction. This alleviates calcineurin inhibition causing anincrease in translocation of NFAT and IL-2 production. CaN, calcineurin.

FIG. 5 depicts that treatment with NU7441 does not reduce cell viabilityin PBMC cells.

FIG. 6A, FIG. 6B, and FIG. 6C depict loss of DNA-PKcs activity reducesrejection in an allogeneic murine skin graft mode, (FIG. 6A) control and(FIG. 6B) DNA-PK inhibitor. (FIG. 6C) decreased rejection withDNA-PK(cs) inhibitor.

FIG. 7A and FIG. 7B depict DNA-PKcs inhibition does not affect PD-1expression. (FIG. 7A) depicts PD1-expression in presence of NU7441 vs.FK506. (FIG. 7B) depicts IL-2 expression in presence of NU7441 vs.FK506.

FIG. 8A and FIG. 8B depict DNA-PKcs inhibition promotes Th1differentiation. qPCR analysis was performed on markers specific to Th1cells following differentiation of naïve CD4+ T cells isolated frommouse spleens into the Th1 T cell subtype. (FIG. 8A): T-Bet, (FIG. 8B):Lymphotoxin.

FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, and FIG. 9E depict DNA-PKcsinhibition blocks Th17 differentiation. qPCR analysis was performed onmarkers specific to Th17 cells following differentiation of naïve CD4+ Tcells isolated from mouse spleens into the Th17 T cell subtype. (FIG.9A): Batf3, (FIG. 9B): RoRyt, (FIG. 9C): IL22, (FIG. 9D): TGFBeta, (FIG.9E): IL17.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compositions and methods for improving thetransplantation outcome and reducing immune response in a subject.

Additional aspects of the invention are described below.

(I) Compositions

One aspect of the present disclosure encompasses a compositioncomprising at least one DNA-PK inhibitor. In some embodiments, theDNA-PK may be inhibited by a nucleotide, an antibody, or a smallmolecule inhibitor.

A composition of the present disclosure may optionally comprise one ormore additional drug(s) or therapeutically active agent(s) in additionto the DNA-PK inhibitor. A composition of the invention may furthercomprise a pharmaceutically acceptable excipient, carrier, or diluent.Further, a composition of the invention may contain preserving agents,solubilizing agents, stabilizing agents, wetting agents, emulsifiers,sweeteners, colorants, odorants, salts (substances of the presentinvention may themselves be provided in the form of a pharmaceuticallyacceptable salt), buffers, coating agents, or antioxidants.

Other aspects of the invention are described in further detail below.

(a) Nucleotide and Antibody Inhibitors

In general, the DNA-PK inhibitor may comprise a nucleotide inhibitor oran antibody inhibitor. In some embodiments, the DNA-PK inhibitor maycomprise a nucleotide inhibitor. In other embodiments, DNA-PK inhibitormay comprise an antibody inhibitor.

(i) Nucleotide Inhibitor

In general, a nucleotide DNA-PK inhibitor may be an RNA sequence. TheRNA sequence may be a short-hairpin RNA (shRNA). The shRNA may be usedto reduce expression of DNA-PK. In an embodiment, the sequence of theshRNA may comprise GCGACATATTATGGAAGAATT (SEQ ID NO: 6);CCACCCAACAACAATATGATT (SEQ ID NO: 7); or GCCATACAAATGTGGAATTAA (SEQ IDNO: 8).

Without being bound by theory, it is believed that the shRNA bind DNA-PKnucleotides which encode the LRR (leucine rich region) motif betweenamino acid resides 1503-1602 of DNA-PK and corresponds to a DNA bindingdomain. (see, for instance, Nucleic Acids Res. 2005 Dec. 9;33(22):6972-81.)

(ii) Antibody Inhibitor

In general, an antibody inhibitor may be an anti-DNA-PK antibody.

Examples of anti-DNA-PK antibodies may include, without limit, ab174576,ab18192, ab32566, ab124918, ab18356, ab230, ab4194, ab4194, ab168854,ab70250, ab44815, ab69527, ab174575, ab133516, ab195537, ab133441,ab97611, and ab218129. These antibodies are commercially available. Inother embodiments, an antibody inhibitor may be a single chain antibody,chimeric antibody, or humanized antibody that comprises the sequenceresponsible for the binding specificity of ab174576, ab18192, ab32566,ab124918, ab18356, ab230, ab4194, ab4194, ab168854, ab70250, ab44815,ab69527, ab174575, ab133516, ab195537, ab133441, ab97611, or ab218129.

(b) Small Molecules

In general, a DNA-PK inhibitor may comprise a small molecule inhibitor.The small molecule inhibitors detailed herein include compounds thatinhibit DNA-PK. Compounds known to inhibit DNA-PK are known in the art.See for example, U.S. Pat. Nos. 9,376,448; 9,592,232; 8,242,115;7,179,912; US 2013/0109687; WO 2011/137428; U.S. Pat. No. 8,404,681; andUS 2008/0090782; the disclosures of which are hereby incorporated byreference. The DNA-PK inhibitors described herein inhibit an activity ofa DNA-PK polypeptide by a percentage of inhibition. Further, the DNA-PKinhibitors inhibit the DNA-PK polypeptide by 50%, by 60%, by 70%, or by80%. The percentage of inhibition may be determined by an in vitrobiochemical assay, an in vitro cell-based assay, or in an in vivo assay.Additionally assays are known to those of skill in the art.

In an embodiment, the small molecule inhibitor may be

or an analog thereof.

In other embodiments, the small molecule inhibitor may be selected fromthe group consisting of

(II) Pharmaceutical Compositions

Another aspect of the present disclosure provides pharmaceuticalcompositions. The pharmaceutical compositions comprise at least oneDNA-PK inhibitor and at least one pharmaceutical acceptable excipient.

(a) Composition

The pharmaceutically acceptable excipient may be a diluent, a binder, afiller, a buffering agent, a pH modifying agent, a disintegrant, adispersant, a preservative, a lubricant, taste-masking agent, aflavoring agent, or a coloring agent. The amount and types of excipientsutilized to form pharmaceutical compositions may be selected accordingto known principles of pharmaceutical science.

(i) Diluent

In one embodiment, the excipient may be a diluent. The diluent may becompressible (i.e., plastically deformable) or abrasively brittle.Non-limiting examples of suitable compressible diluents includemicrocrystalline cellulose (MCC), cellulose derivatives, cellulosepowder, cellulose esters (i.e., acetate and butyrate mixed esters),ethyl cellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, sodium carboxymethylcellulose, cornstarch, phosphated corn starch, pregelatinized corn starch, rice starch,potato starch, tapioca starch, starch-lactose, starch-calcium carbonate,sodium starch glycolate, glucose, fructose, lactose, lactosemonohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol,xylitol, maltodextrin, and trehalose. Non-limiting examples of suitableabrasively brittle diluents include dibasic calcium phosphate (anhydrousor dihydrate), calcium phosphate tribasic, calcium carbonate, andmagnesium carbonate.

(ii) Binder

In another embodiment, the excipient may be a binder. Suitable bindersinclude, but are not limited to, starches, pregelatinized starches,gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylamides,polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohol,polyethylene glycol, polyols, saccharides, oligosaccharides,polypeptides, oligopeptides, and combinations thereof.

(iii) Filler

In another embodiment, the excipient may be a filler. Suitable fillersinclude, but are not limited to, carbohydrates, inorganic compounds, andpolyvinylpyrrolidone. By way of non-limiting example, the filler may becalcium sulfate, both di- and tri-basic, starch, calcium carbonate,magnesium carbonate, microcrystalline cellulose, dibasic calciumphosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc,modified starches, lactose, sucrose, mannitol, or sorbitol.

(iv) Buffering Agent

In still another embodiment, the excipient may be a buffering agent.Representative examples of suitable buffering agents include, but arenot limited to, phosphates, carbonates, citrates, tris buffers, andbuffered saline salts (e.g., Tris buffered saline or phosphate bufferedsaline).

(v) pH Modifier

In various embodiments, the excipient may be a pH modifier. By way ofnon-limiting example, the pH modifying agent may be sodium carbonate,sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

(vi) Disintegrant

In a further embodiment, the excipient may be a disintegrant. Thedisintegrant may be non-effervescent or effervescent. Suitable examplesof non-effervescent disintegrants include, but are not limited to,starches such as corn starch, potato starch, pregelatinized and modifiedstarches thereof, sweeteners, clays, such as bentonite,micro-crystalline cellulose, alginates, sodium starch glycolate, gumssuch as agar, guar, locust bean, karaya, pecitin, and tragacanth.Non-limiting examples of suitable effervescent disintegrants includesodium bicarbonate in combination with citric acid and sodiumbicarbonate in combination with tartaric acid.

(vii) Dispersant

In yet another embodiment, the excipient may be a dispersant ordispersing enhancing agent. Suitable dispersants may include, but arenot limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum,kaolin, bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose.

(viii) Excipient

In another alternate embodiment, the excipient may be a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate,citric acid, sodium citrate; chelators such as EDTA or EGTA; andantimicrobials, such as parabens, chlorobutanol, or phenol.

(ix) Lubricant

In a further embodiment, the excipient may be a lubricant. Non-limitingexamples of suitable lubricants include minerals such as talc or silica;and fats such as vegetable stearin, magnesium stearate, or stearic acid.

(x) Taste-Masking Agent

In yet another embodiment, the excipient may be a taste-masking agent.Taste-masking materials include cellulose ethers; polyethylene glycols;polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers;monoglycerides or triglycerides; acrylic polymers; mixtures of acrylicpolymers with cellulose ethers; cellulose acetate phthalate; andcombinations thereof.

(xi) Flavoring Agent

In an alternate embodiment, the excipient may be a flavoring agent.Flavoring agents may be chosen from synthetic flavor oils and flavoringaromatics and/or natural oils, extracts from plants, leaves, flowers,fruits, and combinations thereof.

(xii) Coloring Agent

In still a further embodiment, the excipient may be a coloring agent.Suitable color additives include, but are not limited to, food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drugand cosmetic colors (Ext. D&C).

The weight fraction of the excipient or combination of excipients in thecomposition may be about 99% or less, about 97% or less, about 95% orless, about 90% or less, about 85% or less, about 80% or less, about 75%or less, about 70% or less, about 65% or less, about 60% or less, about55% or less, about 50% or less, about 45% or less, about 40% or less,about 35% or less, about 30% or less, about 25% or less, about 20% orless, about 15% or less, about 10% or less, about 5% or less, about 2%,or about 1% or less of the total weight of the composition.

(b) Administration

(i) Dosage Forms

The composition can be formulated into various dosage forms andadministered by a number of different means that will deliver atherapeutically effective amount of the active ingredient. Suchcompositions can be administered orally (e.g. inhalation), parenterally,or topically in dosage unit formulations containing conventionalnontoxic pharmaceutically acceptable carriers, adjuvants, and vehiclesas desired. Topical administration may also involve the use oftransdermal administration such as transdermal patches or iontophoresisdevices. The term parenteral as used herein includes subcutaneous,intravenous, intramuscular, intra-articular, or intrasternal injection,or infusion techniques. Formulation of drugs is discussed in, forexample, Gennaro, A. R., Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa. (18^(th) ed, 1995), and Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., NewYork, N.Y. (1980). In a specific embodiment, a composition may be a foodsupplement or a composition may be a cosmetic.

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, powders, pellets, and granules. In such solid dosageforms, the active ingredient is ordinarily combined with one or morepharmaceutically acceptable excipients, examples of which are detailedabove. Oral preparations may also be administered as aqueoussuspensions, elixirs, or syrups. For these, the active ingredient may becombined with various sweetening or flavoring agents, coloring agents,and, if so desired, emulsifying and/or suspending agents, as well asdiluents such as water, ethanol, glycerin, and combinations thereof. Foradministration by inhalation, the compounds are delivered in the form ofan aerosol spray from pressured container or dispenser which contains asuitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

For parenteral administration (including subcutaneous, intradermal,intravenous, intramuscular, intra-articular and intraperitoneal), thepreparation may be an aqueous or an oil-based solution. Aqueoussolutions may include a sterile diluent such as water, saline solution,a pharmaceutically acceptable polyol such as glycerol, propylene glycol,or other synthetic solvents; an antibacterial and/or antifungal agentsuch as benzyl alcohol, methyl paraben, chlorobutanol, phenol,thimerosal, and the like; an antioxidant such as ascorbic acid or sodiumbisulfite; a chelating agent such as etheylenediaminetetraacetic acid; abuffer such as acetate, citrate, or phosphate; and/or an agent for theadjustment of tonicity such as sodium chloride, dextrose, or apolyalcohol such as mannitol or sorbitol. The pH of the aqueous solutionmay be adjusted with acids or bases such as hydrochloric acid or sodiumhydroxide. Oil-based solutions or suspensions may further comprisesesame, peanut, olive oil, or mineral oil. The compositions may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carried, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets.

For topical (e.g., transdermal or transmucosal) administration,penetrants appropriate to the barrier to be permeated are generallyincluded in the preparation. Pharmaceutical compositions adapted fortopical administration may be formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays,aerosols, or oils. In some embodiments, the pharmaceutical compositionis applied as a topical ointment or cream. When formulated in anointment, the active ingredient may be employed with either a paraffinicor a water-miscible ointment base. Alternatively, the active ingredientmay be formulated in a cream with an oil-in-water cream base or awater-in-oil base. Pharmaceutical compositions adapted for topicaladministration to the eye include eye drops wherein the activeingredient is dissolved or suspended in a suitable carrier, especiallyan aqueous solvent. Pharmaceutical compositions adapted for topicaladministration in the mouth include lozenges, pastilles, and mouthwashes. Transmucosal administration may be accomplished through the useof nasal sprays, aerosol sprays, tablets, or suppositories, andtransdermal administration may be via ointments, salves, gels, patches,or creams as generally known in the art.

In certain embodiments, a composition comprising at least one DNA-PKinhibitor is encapsulated in a suitable vehicle to either aid in thedelivery of the compound to target cells, to increase the stability ofthe composition, or to minimize potential toxicity of the composition.As will be appreciated by a skilled artisan, a variety of vehicles aresuitable for delivering a composition of the present invention.Non-limiting examples of suitable structured fluid delivery systems mayinclude nanoparticles, liposomes, microemulsions, micelles, dendrimers,and other phospholipid-containing systems. Methods of incorporatingcompositions into delivery vehicles are known in the art.

In one alternative embodiment, a liposome delivery vehicle may beutilized. Liposomes, depending upon the embodiment, are suitable fordelivery of at least one DNA-PK inhibitor in view of their structuraland chemical properties. Generally speaking, liposomes are sphericalvesicles with a phospholipid bilayer membrane. The lipid bilayer of aliposome may fuse with other bilayers (e.g., the cell membrane), thusdelivering the contents of the liposome to cells. In this manner, atleast one DNA-PK inhibitor may be selectively delivered to a cell byencapsulation in a liposome that fuses with the targeted cell'smembrane.

Liposomes may be comprised of a variety of different types ofphosolipids having varying hydrocarbon chain lengths. Phospholipidsgenerally comprise two fatty acids linked through glycerol phosphate toone of a variety of polar groups. Suitable phospholids includephosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol(PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG),phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fattyacid chains comprising the phospholipids may range from about 6 to about26 carbon atoms in length, and the lipid chains may be saturated orunsaturated. Suitable fatty acid chains include (common name presentedin parentheses) n-dodecanoate (laurate), n-tretradecanoate (myristate),n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate(arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate),cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate),cis,cis-9,12-octadecandienoate (linoleate), all cis-9, 12,15-octadecatrienoate (linolenate), and allcis-5,8,11,14-eicosatetraenoate (arachidonate). The two fatty acidchains of a phospholipid may be identical or different. Acceptablephospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS,distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl,oleoyl PS, palmitoyl, linolenyl PS, and the like.

The phospholipids may come from any natural source, and, as such, maycomprise a mixture of phospholipids. For example, egg yolk is rich inPC, PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brainor spinal cord is enriched in PS. Phospholipids may come from syntheticsources too. Mixtures of phospholipids having a varied ratio ofindividual phospholipids may be used. Mixtures of differentphospholipids may result in liposome compositions having advantageousactivity or stability of activity properties. The above mentionedphospholipids may be mixed, in optimal ratios with cationic lipids, suchas N-(1-(2,3-dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride,1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,3,3′-deheptyloxacarbocyanine iodide,1,1′-dedodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,1,1′-dioleyl-3,3,3′,3′-tetramethylindo carbocyanine methanesulfonate,N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or1,1,-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate.

Liposomes may optionally comprise sphingolipids, in which spingosine isthe structural counterpart of glycerol and one of the one fatty acids ofa phosphoglyceride, or cholesterol, a major component of animal cellmembranes. Liposomes may optionally contain pegylated lipids, which arelipids covalently linked to polymers of polyethylene glycol (PEG). PEGsmay range in size from about 500 to about 10,000 daltons.

Liposomes may further comprise a suitable solvent. The solvent may be anorganic solvent or an inorganic solvent. Suitable solvents include, butare not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone,N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide,tetrahydrofuran, or combinations thereof.

Liposomes carrying at least one DNA-PK inhibitor may be prepared by anyknown method of preparing liposomes for drug delivery, such as, forexample, detailed in U.S. Pat. Nos. 4,241,046; 4,394,448; 4,529,561;4,755,388; 4,828,837; 4,925,661; 4,954,345; 4,957,735; 5,043,164;5,064,655; 5,077,211; and 5,264,618, the disclosures of which are herebyincorporated by reference in their entirety. For example, liposomes maybe prepared by sonicating lipids in an aqueous solution, solventinjection, lipid hydration, reverse evaporation, or freeze drying byrepeated freezing and thawing. In a preferred embodiment the liposomesare formed by sonication. The liposomes may be multilamellar, which havemany layers like an onion, or unilamellar. The liposomes may be large orsmall. Continued high-shear sonication tends to form smaller unilamellarliposomes.

As would be apparent to one of ordinary skill, all of the parametersthat govern liposome formation may be varied. These parameters include,but are not limited to, temperature, pH, concentration of the DNA-PKinhibitor, concentration and composition of lipid, concentration ofmultivalent cations, rate of mixing, presence of and concentration ofsolvent.

In another embodiment, a composition of the invention may be deliveredto a cell as a microemulsion. Microemulsions are generally clear,thermodynamically stable solutions comprising an aqueous solution, asurfactant, and “oil.” The “oil” in this case, is the supercriticalfluid phase. The surfactant rests at the oil-water interface. Any of avariety of surfactants are suitable for use in microemulsionformulations including those described herein or otherwise known in theart. The aqueous microdomains suitable for use in the inventiongenerally will have characteristic structural dimensions from about 5 nmto about 100 nm. Aggregates of this size are poor scatterers of visiblelight and hence, these solutions are optically clear. As will beappreciated by a skilled artisan, microemulsions can and will have amultitude of different microscopic structures including sphere, rod, ordisc shaped aggregates. In one embodiment, the structure may bemicelles, which are the simplest microemulsion structures that aregenerally spherical or cylindrical objects. Micelles are like drops ofoil in water, and reverse micelles are like drops of water in oil. In analternative embodiment, the microemulsion structure is the lamellae. Itcomprises consecutive layers of water and oil separated by layers ofsurfactant. The “oil” of microemulsions optimally comprisesphospholipids. Any of the phospholipids detailed above for liposomes aresuitable for embodiments directed to microemulsions. At least one DNA-PKinhibitor may be encapsulated in a microemulsion by any method generallyknown in the art.

In yet another embodiment, at least one DNA-PK inhibitor may bedelivered in a dendritic macromolecule, or a dendrimer. Generallyspeaking, a dendrimer is a branched tree-like molecule, in which eachbranch is an interlinked chain of molecules that divides into two newbranches (molecules) after a certain length. This branching continuesuntil the branches (molecules) become so densely packed that the canopyforms a globe. Generally, the properties of dendrimers are determined bythe functional groups at their surface. For example, hydrophilic endgroups, such as carboxyl groups, would typically make a water-solubledendrimer. Alternatively, phospholipids may be incorporated in thesurface of a dendrimer to facilitate absorption across the skin. Any ofthe phospholipids detailed for use in liposome embodiments are suitablefor use in dendrimer embodiments. Any method generally known in the artmay be utilized to make dendrimers and to encapsulate compositions ofthe invention therein. For example, dendrimers may be produced by aniterative sequence of reaction steps, in which each additional iterationleads to a higher order dendrimer. Consequently, they have a regular,highly branched 3D structure, with nearly uniform size and shape.Furthermore, the final size of a dendrimer is typically controlled bythe number of iterative steps used during synthesis. A variety ofdendrimer sizes are suitable for use in the invention. Generally, thesize of dendrimers may range from about 1 nm to about 100 nm.

(ii) Dosage

Dosages of the pharmaceutical compositions can vary between wide limits,depending upon the disease or disorder to be treated, the age of thesubject, and the condition of the subject to be treated. In anembodiment, the amount of the DNA-PK inhibitor in the pharmaceuticalcomposition is an amount to effectively inhibit DNA-PK.

(iii) Subject

A subject may be a rodent, a human, a livestock animal, a companionanimal, or a zoological animal. In one embodiment, the subject may be arodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment,the subject may be a livestock animal. Non-limiting examples of suitablelivestock animals may include pigs, cows, horses, goats, sheep, llamas,and alpacas. In still another embodiment, the subject may be a companionanimal. Non-limiting examples of companion animals may include pets suchas dogs, cats, rabbits, and birds. In yet another embodiment, thesubject may be a zoological animal. As used herein, a “zoologicalanimal” refers to an animal that may be found in a zoo. Such animals mayinclude non-human primates, large cats, wolves, and bears. In apreferred embodiment, the subject is a human.

(III) Methods

In an aspect, the present disclosure provides methods of reducing IL-2secretion in T cells. The method comprises contacting a T cell with acomposition comprising a DNA-PK inhibitor of the present invention.Suitable DNA-PK inhibitors are disclosed herein, for instance thosedescribed in Section I. In some embodiments, the DNA-PK inhibitor in thepresent invention inhibits production of IL-2 by activated T cellscompared to activated T-cells which have not been contacted with aDNA-PK inhibitor. IL-2 expression and/or secretion may be measuredaccording to standard methods known in the art, including thosedescribed in the Examples.

Contacting the T cell with a DNA-PK inhibitor composition may occur invitro, in vivo, or ex vivo. For example, in one aspect, the inventionprovides contacting a cell culture comprising T-cells with a compositioncomprising a DNA-PK inhibitor of the invention. In another aspect, theinvention provides a method of contacting T-cells in a subject byadministering a composition comprising a DNA-PK inhibitor of theinvention.

An additional aspect is a method for treating a disease or disordercaused by IL-2 production or acceleration of IL-2 receptor expression.In this aspect, a DNA-PK inhibitor is administered to a subject forprophylaxis or treatment of a disease or disorder caused by IL-2production or acceleration of IL-2 receptor expression. The IL-2 relateddiseases in the present invention are diseases caused by IL-2 productionor acceleration of IL-2 receptor expression. By way of non-limitingexample, IL-2 related diseases may include AIDS, skin diseases(psoriasis, atopic dermatitis, urticaria), internal diseases (lupusnephritis), ophthalmic diseases (allergic conjunctivitis, sty,chalazion, spring catarrh, uveitis, cancer), autoimmune diseases(polymyositis, Hashimoto's disease, Behcet's disease, ankylosingspondylitis, systemic sclerosis, Sjogren's syndrome, pollenosis,scleroderma), gastrointestinal diseases, inflammatory diseases (gout,psoriatic arthritis, rheumatoid arthritis), central nervous systemdiseases (multiple sclerosis), respiratory diseases (asthma, chronicobstructive pulmonary disease), fibromyalgia, myasthenia gravis,sarcoidosis, nasal inflammation, and nasal catarrh.

Another aspect of the present disclosure encompasses a method ofreducing an immune response in a subject. The method comprisesadministering to a subject an effective amount of a compositioncomprising a DNA-PK inhibitor. In some embodiments, the compositionreduces an immune response in a subject by reducing cellular and/orhumoral immunity in the subject. As used herein, the term “immuneresponse” includes T cell mediated and/or B cell mediated immuneresponses. Non-limiting exemplary immune responses include T cellresponses, e.g., cytokine production, and cellular cytotoxicity. Inaddition, the term immune response includes immune responses that areindirectly effected by T cell activation, e.g., antibody production(humoral responses) and activation of cytokine responsive cells, e.g.,macrophages.

Reducing an immune response can be in the form of inhibiting ordown-regulating an immune response already in progress or may involvepreventing the induction of an immune response. For example,administration of a composition comprising a DNA-PK inhibitor to asubject may reduce cytokine production, cellular toxicity, antibodyproduction or the activation of cytokine response cells compared to acontrol subject who has who has not been contacted with the composition.In another aspect, administration of a composition comprising a DNA-PKinhibitor may reduce cytokine production, cellular toxicity, antibodyproduction or the activation of cytokine response cells compared to thesame subject prior to administration of the DNA-PK inhibitor.

In yet another aspect, the present disclosure provides a method toimprove the transplantation outcome in a subject receiving an organ ortissue transplant. In one aspect, the invention provides a method ofreducing an immune response in a subject receiving an organ or tissuetransplant. In another aspect, the present invention provides reducingIL-2 secretion in T-cells of a subject receiving an organ or tissuetransplant. The methods comprise administering to a subject receiving anorgan or tissue transplant an effective amount of a compositioncomprising a DNA-PK inhibitor. In the various embodiments,administration may occur prior to transplantation, duringtransplantation or post-transplant.

In non-limiting examples, the transplant recipients may be recipients ofkidney, liver, heart, heart-lung, bone-marrow, and cornea transplants.As used herein, the term “transplantation” refers to the process oftaking a cell, tissue, or organ, called a “transplant” or “graft” fromone individual and placing it or them into a (usually) differentindividual. The individual who provides the transplant is called the“donor” and the individual who received the transplant is called the“host” (or “recipient”). An organ, or graft, transplanted between twogenetically different individuals of the same species is called an“allograft”. A graft transplanted between individuals of differentspecies is called a “xenograft”. The organ transplant tissue itself istypically human in origin, but may also be from another species such asthe rhesus monkey.

In accordance with the invention, in some embodiments, transplantrecipients have improved transplantation outcomes including reducedtransplant rejection in a subject. As used herein, “transplantrejection” is characterized by an acute or chronic diminuation in thephysiological function of a transplanted organ. Acute rejectiontypically occurs within the first year post transplantation andgenerally speaking is a consequence of cell-mediated immune response(e.g. T cells). Chronic rejection occurs several years followingtransplant resulting from antibody-mediated immune response (e.g. Bcells). In some aspects, reduced transplant rejection in transplantedorgan or tissue function is measured by biological factors specific tothe organ transplanted. For example, for kidney transplant rejectionassessment, decreased glomerular atrophy, reduced intimal thickening,reduced tubular atrophy, reduced interstitial fibrosis, reducedlymphocyte infiltration and reduced cortical scarring independently ortaken together are indicators of reduced graft rejection. Similarly, forheart transplant reduced rejection assessment includes, reduced cardiacvessel disease post-transplant, and reduced graft intimal hyperplasiaindependently or taken together are indicators of reduced graftrejection. In non-limiting examples, reduced transplant rejectiontreatment is assessed in accordance with the present invention by one ormore of the following organ-dependent parameters: decreased coronarygraft intimal hyperplasia compared to grafted vessels in a subject notreceiving a DNA-PK inhibitor; improved renal function as measured byserial serum creatinine levels; graft survival prolongation;hyalinization and cortical scarring in renal grafts; decreasedlymphocytic infiltration, vasculitis, infarction, ischemic, thrombosis,intimal thickening, glomerular atrophy, glomerular sclerosis, tubularatrophy, hyalinization, interstitial fibrosis, cortical fibrosis, serumcreatinine levels, intimal proliferation, hypertrophy, cardiac vesseldisease post-transplant, graft intimal hyperplasia, luminal occlusion,or bronchitis obliterans. It is understood that the biological factorswhich can be measured as an indicator of reduced transplant rejectionare specific to the organ transplanted and are understood by thoseskilled in the art.

In some embodiments, the methods of the invention provide a DNA-PKinhibitor of the present invention used in combination with one or moreof a nonsteroidal anti-inflammatory agent, a steroidal anti-inflammatoryagent, an immune suppressant, an antihistamine, an antirheumatic drugand a biological preparation such as infliximab, adalimumab,tocilizumab, etc. Non-limiting examples of suitable nonsteroidalanti-inflammatory agents may include indomethacin, ibuprofen,diclofenac, and aspirin. Non-limiting examples of suitable steroidalanti-inflammatory agents may include dexamethasone, betamethasone,prednisolone, and triamcinolone. Non-limiting examples of suitableimmunosuppressants may include tacrolimus, cyclosporine, and sirolimus.Non-limiting examples of suitable antihistamines may includediphenhydramine, chlorpheniramine, triprolidine, promethazine,alimemazine, hydroxyzine, cyproheptadine, fexofenadine, olopatadine,epinastine, loratadine, cetirizine, bepotastine, and mequitazine.Non-limiting examples of suitable antirheumatic drugs may includebucillamine, salazosulfapyridine, and methotrexate.

(a) Administration

In certain aspects, a therapeutically effective amount of a compositionof the invention may be administered to a subject. Administration isperformed using standard effective techniques. In a preferredembodiment, a composition is administered orally, parenterally, ortopically.

For therapeutic applications, a therapeutically effective amount of acomposition of the invention is administered to a subject. A“therapeutically effective amount” is an amount of the therapeuticcomposition sufficient to produce a measurable response (e.g., decreasedIL-2 expression, decreased organ transplant rejection, graft survivalprolongation, and the like). Actual dosage levels of active ingredientsin a therapeutic composition of the invention can be varied so as toadminister an amount of the active compound(s) that is effective toachieve the desired therapeutic response for a particular subject. Theselected dosage level will depend upon a variety of factors includingthe activity of the therapeutic composition, formulation, the route ofadministration, combination with other drugs or treatments, age, diseaseor condition, the organ or tissue transplanted, the symptoms, and thephysical condition and prior medical history of the subject beingtreated. In some embodiments, a minimal dose is administered, and doseis escalated in the absence of dose-limiting toxicity. Determination andadjustment of a therapeutically effective dose, as well as evaluation ofwhen and how to make such adjustments, are known to those of ordinaryskill in the art of medicine.

The timing of administration of the treatment is under stood to berelative to the timing of the transplantation or to disease itself andduration of treatment will be determined by the circumstancessurrounding the case. For example, treatment may occur prior totransplantation or post-transplant. Treatment could begin in a hospitalor clinic itself, or at a later time after discharge from the hospitalor after being seen in an outpatient clinic.

Duration of treatment could range from a single dose administered on aone-time basis to a life-long course of therapeutic treatments. Theduration of treatment can and will vary depending on the subject and thedisease or disorder to be treated. For example, the duration oftreatment may be for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7days. Or, the duration of treatment may be for 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks or 6 weeks. Alternatively, the duration of treatmentmay be for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 12 months. In stillanother embodiment, the duration of treatment may be for 1 year, 2years, 3 years, 4 years, 5 years, or greater than 5 years. It is alsocontemplated that administration may be frequent for a period of timeand then administration may be spaced out for a period of time. Forexample, duration of treatment may be 5 days, then no treatment for 9days, then treatment for 5 days.

The frequency of dosing may be once, twice, three times or more daily oronce, twice, three times or more per week or per month, or as needed asto effectively treat the symptoms or disease. In certain embodiments,the frequency of dosing may be once, twice or three times daily. Forexample, a dose may be administered every 24 hours, every 12 hours, orevery 8 hours. In other embodiments, the frequency of dosing may beonce, twice or three times weekly. For example, a dose may beadministered every 2 days, every 3 days or every 4 days. In a differentembodiment, the frequency of dosing may be one, twice, three or fourtimes monthly. For example, a dose may be administered every 1 week,every 2 weeks, every 3 weeks or every 4 weeks.

(b) Subject

A subject may be a rodent, a human, a livestock animal, a companionanimal, or a zoological animal. In one embodiment, the subject may be arodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment,the subject may be a livestock animal. Non-limiting examples of suitablelivestock animals may include pigs, cows, horses, goats, sheep, llamasand alpacas. In still another embodiment, the subject may be a companionanimal. Non-limiting examples of companion animals may include pets suchas dogs, cats, rabbits, and birds. In yet another embodiment, thesubject may be a zoological animal. As used herein, a “zoologicalanimal” refers to an animal that may be found in a zoo. Such animals mayinclude non-human primates, large cats, wolves, and bears. In apreferred embodiment, the subject is a human.

The human subject may be of any age. In some embodiments, the humansubject may be about 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 years of age or older. In some preferred embodiments, the humansubject is 30 years of age or older. In other preferred embodiments, thehuman subject is 40 years of age or older. In other preferredembodiments, the human subject is 45 years of age or older. In yet otherpreferred embodiments, the human subject is 50 years of age or older. Instill other preferred embodiments, the human subject is 55 years of ageor older. In other preferred embodiments, the human subject is 60 yearsof age or older. In yet other preferred embodiments, the human subjectis 65 years of age or older. In still other preferred embodiments, thehuman subject is 70 years of age or older. In other preferredembodiments, the human subject is 75 years of age or older. In stillother preferred embodiments, the human subject is 80 years of age orolder. In yet other preferred embodiments, the human subject is 85 yearsof age or older. In still other preferred embodiments, the human subjectis 90 years of age or older.

Definitions

When introducing elements of the present disclosure or the preferredaspects(s) thereof, the articles “a,” “an,” “the,” and “said” areintended to mean that there are one or more of the elements. The terms“comprising,” “including,” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

The term “therapeutically effective amount” means the dose needed toeffectively treat the physiological effects of graft rejection.

“IL-2” (Interleukin-2) is a cytokine produced mainly by activated Tcells, and acts on the cells such as T cells, B cells, macrophages, etc.IL-2 promotes proliferation and activation of T cells, proliferation andacceleration of the antibody-producing ability of B cells, activation ofmonocytes and macrophages, proliferation and activation of naturalkiller cells (NK cell), and inducing action of lymphokine-activatedkiller cells, etc.

As used herein, the term “immune cell” includes cells that are ofhematopoietic origin and that play a role in the immune response. Immunecells include lymphocytes, such as B cells and T cells; natural killercells; myeloid cells, such as monocytes, macrophages, eosinophils, mastcells, basophils, and granulocytes.

As used herein, the term “T cell” includes CD4+ T cells and CD8+ Tcells. The term T cell also includes both T helper 1 type T cells and Thelper 2 type T cells. The term “antigen presenting cell” includesprofessional antigen presenting cells (e.g., B lymphocytes, monocytes,dendritic cells, Langerhans cells) as well as other antigen presentingcells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes).

The term “antibody” as used herein also includes an “antigen-bindingportion” of an antibody (or simply “antibody portion”). The term“antigen-binding portion”, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., DNA-PK). It has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778).Such single chain antibodies are also intended to be encompassed withinthe term “antigen-binding portion” of an antibody. Any VH and VLsequences of specific scFv can be linked to human immunoglobulinconstant region cDNA or genomic sequences, in order to generateexpression vectors encoding complete IgG molecules or other isotypes. VHand V1 can also be used in the generation of Fab, Fv or other fragmentsof immunoglobulins using either protein chemistry or recombinant DNAtechnology. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g. Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

As various changes could be made in the above-described materials andmethods without departing from the scope of the invention, it isintended that all matter contained in the above description and in theexamples given below, shall be interpreted as illustrative and not in alimiting sense.

EXAMPLES

The following examples are included to demonstrate various embodimentsof the present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1: DNA-PKcs Controls Calcineurin Mediated IL-2 Production in TLymphocytes Introduction

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a460 kDa polypeptide member of the PI3k family. It was initiallydiscovered to be a key component in non-homologous end-joining (NHEJ)which is the predominant pathway used to repair DNA double strand breaksin mammalian cells and is critical for V(D)J recombination [1, 2].DNA-PKcs is believed to serve as a recruiting and scaffolding proteinfor DNA ligase [3]. Knock out of DNA-PKcs activity in mammals results ina Severe Combined Immunodeficiency (SCID) phenotype which ischaracterized by diminished levels of mature B and T cells [4-6]. Thishas been attributed to disruption of V(D)J recombination which isnecessary for lymphocyte development and responsible for both antibodyand T cell receptor diversity [7, 8]. Without being bound by theory, itis thought that this enzyme is involved in other aspects of the immuneresponse including Interleukin-2 (IL-2) signaling since the disruptionof the IL-2 pathway in IL-2 receptor mutants also results in a SCIDphenotype [9]. Of note, DNA-PKcs has previously been associated withmultiple receptor signaling pathways including EGF, RET, and the insulinsignaling pathway and phosphorylates key molecules associated with cellgrowth, e.g., AKT [10-14].

Well described, IL-2 is a T cell-derived cytokine that influences amultitude of key elements in the immune response including theproliferation and differentiation of B and T lymphocytes [15].Expression of IL-2 is initiated upon calcineurin activation. Calcineurinis a calcium and calmodulin-dependent protein serine/threoninephosphatase that upon activation, dephosphorylates Nuclear Factor ofActivated T-cells (NFAT) allowing it to translocate to the nucleus andupregulate expression of target genes (including IL-2) [15-17]. IL-2then binds to its receptor IL-2R, expressed on the surface oflymphocytes, to induce signaling that impacts both arms of the immuneresponse, humoral and cellular immunity [18]. IL-2 is known to promotethe expansion and maturation of B and T lymphocytes and regulates thedifferentiation of T cells into effector or regulatory T cells [15-17].

To evaluate the function of DNA-PKcs in this pathway, its activity wasinhibited by either shRNA or the commercially available inhibitor NU7441in Jurkat cells, a human T cell line, and the effect on IL-2 levels wasanalyzed. Inhibiting DNA-PKcs in activated Jurkat cells resulted inreduced calcineurin activity, loss of NFAT translocation to the nucleusand decreased IL-2 expression. It was showed that this effect was linkedto the calcineurin inhibitor, Cabin1. Cabin1 directly binds to activatedcalcineurin and blocks its dephosphorylation of NFAT. Overexpressingfull length Cabin1 or its N-terminal region in Jurkat cells has beenshown to reduce IL-2 expression by inhibiting the calcineurin-NFATpathway [19, 20]. Cabin1 was also identified to function in DNA damageby inhibiting activity of p53 [21]. Through these studies it wasrevealed that phosphorylation of Cabin1 by the checkpoint kinase CHK2targets it for ubiquination and degradation [22]. Interestingly,phosphorylation by DNA-PKcs is known to regulate activity of CHK2.DNA-PKcs phosphorylates CHK2 at site Thr68 thereby activating the kinase[23]. It was showed that inhibiting DNA-PKcs in Jurkat cells resulted ina decrease in CHK2 phosphorylation causing an increase in Cabin1expression. This novel pathway for regulation of IL-2 signalingindicates a much broader function for DNA-PKcs in the immune system thanpreviously understood and further explains the development of a SCIDphenotype in mice lacking DNA-PKcs activity.

Methods and Materials

Materials: PHA-L, PMA, X-treme GENE transfection reagent, and 0.1%poly-lysine solution were purchased from Sigma-Aldrich (St. Louis, Mo.).NU7441 was purchased from Selleckchem (Houston, Tex.). shRNA againstDNA-PKcs was purchased from ORIGENE (Rockville, Md.). Dynabeads HumanT-Activator CD3/CD28 was purchased from Thermo Fisher Scientific(Waltham, Mass.).

Cell Culture:

Human peripheral blood mononuclear cells (PBMC,) and Jurkat cells werepurchased from ATCC (PCS-800-011, Manassas, Va.). Cells were maintainedat 37° C. in a humidified atmosphere composed of 5% CO₂. Jurkat cellswere cultured in RPMI 1640 medium which was supplemented with 10% FCSand human PBMC was cultured in RPMI 1640 medium which was supplementedwith 10% FCS and pen/strep. Both Jurkat cells and PBMC were stimulatedwith PHA (50 ng/mL) and PMA (1 μg/mL) for 24 hours prior to harvestingfor IL-2 detection or 6 hours prior for western blot analysis. TheNU7441 DNA-PKcs inhibitor was added at varying concentrations at thetime of stimulation.

Knockdown of DNA-PKcs in Jurkat Cells:

Jurkat cells (5×10⁵ cells/well) were grown in 6-well plates. The cellswere transfected with short-hairpin RNA (shRNA) plasmids generated byOrigene. (2.5 μg of scramble (SEQ ID NO: 5) or 2.5 and 5 μg of specificto DNA-PKcs (SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; and SEQ ID NO:4)) using X-TREME GENE and incubated for 72 hours. Four shRNA plasmidswere obtained from ORIGENE that target various regions of DNA-PKcs. TheshRNA plasmid that provided the best knock down of DNA-PKcs expressionwas used for our experiments. The cells were subjected to Western blotanalysis and IL-2 ELISA assay. shRNA plasmids against DNA-PKcs waspurchased from ORIGENE (Rockville, Md.).

Cell Lysis and Nuclear Extract:

Cells were washed with cold PBS twice and centrifuged at 5000 rpm for 5minutes. For the nuclear extract, pellets were suspended with 800 μL of10 mN HEPES lysis buffer (10 mM HEPES at pH 7.9, 10 mM KCl, 1 mM DTT,and 1× protease and phosphatase inhibitor) and incubated on ice for 15minutes. 50 μL of NP-40 (10% in water) was added and the pellets weremixed for 10 seconds. Lysates were centrifuged for 5 minutes at 4° C. at13,000 rpm. The supernatant solution which is containing the cytosolicfraction was discarded and the pellets were resuspended in 20 mM HEPESlysis buffer (20 mM HEPES at pH 7.9, 0.4 M NaCl, 1 mM DTT, and 1×protease, and phosphatase inhibitor) and incubated on ice for 15 minuteswith intermittent mixing. Lysates were centrifuged for 10 minutes at 4°C. at 13,000 rpm. The supernatant containing nuclear extract was storedat −20° C. until they were used for the Western blot analysis. For totalcell lysates, cell pellets were resuspended with 100 μL of RIPA buffer(150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 50 mM Tris at pH 8.0) andincubated on ice for 10 minutes. The lysates were centrifuged for 10minutes at 4° C. at 13,000 rpm and the supernatant solutions were storedat −20° C. until they were used for the Western blot analysis.

Western Blot Analysis:

Nuclear extract lysates were separated on 3-8% Tris-Acetate gels(Invitrogen). Total cell lysates were separated on 4-20% Tris-Glycinegels (Bio-Rad). Gels were transferred onto PVDF membrane (Millipore) for2 hours in the cold room at 100V. Immunoblotting was performed usingfollowing antibodies: pDNA-PKcs at S2056 (ab18192, Abcam), DNAPK(ab53701, Abcam), pNFAT2 at S237 (ab183023, Abcam), CABIN1 (12660S, Cellsignaling), GAPDH (MAB374, Millipore), and Lam in B1 (ab16048, Abcam).HRP-conjugated secondary antibody anti-Rabbit and anti-Mouse (7074S and7076S, Cell Signaling) was used.

Cell Viability Assay:

Cell viability assay was performed using Promega CELLTITER 96 AQUEOUSOne Solution Cell Proliferation Assay (Madison, Wis.) and following themanufacturer's protocol. Briefly, 100 μL of PBMC or Jurkat cells wereplated in a 96-well plate and treated with various concentration ofNU7441 for 48 hours. CELLTITER solution (20 μL/100 μL of cellsuspension) was added to the cells and the plate was incubated for 3hours at 37° C. and the absorbance at 490 nm was recorded usingSynergyHTX (BioTek, Winooski, Vt.) plate reader.

Immunofluorescence of NFAT:

Jurkat cells were plated in poly-lysine treated 35 mm dish withglass-bottom (BioTek) overnight. The cells were fixed with 4%formaldehyde and permeabilized with permeabilization solution (0.2%TRITON X-100+OVA solution (0.1 mg/mL), 0.01% sodium azide). NFAT wasprobed with anti-NFAT2 (ab2796, Abcam) in permeabilization solutionovernight at 4° C., and then washed with PBS. Fluorescein(FITC)-conjugated AffiniPure Donkey Anti-Mouse IgG (715-095-151, JacksonImmunoResearch) was applied to the cells for 1 hour at room temperature.The cells were washed with PBS and ProLong Antifade reagent with DAPI(Molecular Probes) was applied. All samples were analyzed on an OlympusFluoview FV1000 laser confocal microscope. Images from all microscopyexperiments were processed using the FV10-ASW 3.1 Viewer (Olympus).

Detection of Secreted IL-2:

Secreted IL-2 was detected by Human IL-2 ELISA Kit from ThermoScientific (Waltham, Mass.). The manufacturer's protocol was followed.Prior to harvesting, cells were treated with PHA (50 ng/mL) and PMA (1μg/mL) for 24 hours with or without the NU7441 inhibitor. Jurkat cellsstimulated with the anti-CD28/CD3 dynabeads were done so according tothe manufacturer's protocol at a 1:1 ratio for 24 hours prior toharvesting. After stimulation, supernatant samples of Jurkat cells orPBMCs (2 million cells/mL) were collected and diluted 10 times beforethe assay. IL-2 standards and samples (50 μL) and Biotinylated antibodyreagents (50 μL) were added to each well and the plate was incubated for3 hours at room temperature. The plate was washed 3 times and 100 μL ofStreptavidin-HRP solution was added. After 30 minutes of incubation atroom temperature, the plate was washed 3 times. TMB substrate (100 μL)was added and incubated for 30 minutes in the dark at room temperature.Stop solution was added and the absorbance of each well was read at 450nm using the plate reader.

Detection of Calcineurin and mTOR Activities:

Calcineurin phosphatase activity was detected by Calcineurin CellularActivity Assay Kit from Millipore (Billerica, Mass.) and mTOR signalingwas detected by Calcium Detection Kit from Abcam (Cambridge, Mass.). Themanufacturer's protocols were followed. Briefly, Jurkat cells (2 millioncells/mL) were lysed using lysis buffer. For calcineurin activity, thecell lysates were desalted by gel filtration to remove free phosphatesbefore the assay and were subjected to calcineurin activity assay. Theabsorbance of each sample was read at 620 nm using the plate reader. FormTOR ELISA assay, the cell lysates were added to each well and antibodyagainst phosphorylated mTOR at serine 2448 was used to detect theactivity of mTOR signaling. The absorbance of each sample was read at450 nm using the plate reader.

Measurements of Calcium Ions:

Intracellular concentration of calcium ions was measured by CalciumDetection Assay Kit from Abcam (Cambridge, Mass.). The manufacturer'sprotocols were followed. Briefly, Jurkat cells (2 million cells/mL) werelysed using cold PBS with 0.1% NP-40. The cell lysates were diluted 10times before use. After following the assay protocol, the absorbance ofeach sample was read at 575 nm using the plate reader.

Statistical Analysis:

Assays to monitor IL-2 levels, calcineurin and mTOR activities, and Ca²⁺ion levels were performed in both technical triplicate and biologicaltriplicate. Standard student t-test were performed to compare groupmeans. Means with p-value below 0.05 were considered statisticallydifferent.

Results

DNA-PKcs Regulates IL-2 Secretion in T Cells:

The immune cytokine IL-2 is a key element of the immune responseaffecting both the humoral and cell-mediated arms of the immune system.To determine if DNA-PKcs regulates T cell-mediated IL-2 production, theeffect of the DNA-PKcs inhibitor NU7441 on IL-2 in Jurkat cells wasevaluated. NU7441 is a potent and specific inhibitor of DNA-PKcs whichdoes not interfere with ATR or ATM activation [24]. It was determinedthat NU7441 at varying concentrations did not alter the viability ofJurkat cells (FIG. 1A). Next, the production of IL-2 in Jurkat cellstreated with NU7441 was monitored. In FIG. 1B, the expected spike inIL-2 levels following 24 hour stimulation with PMA+PHA was observed. Inthe presence of the inhibitor, the level of IL-2 was significantlydecreased with 2.5 μM of NU7441 and further decreased with 5 μM (FIG.1B). During an immune response, T cells are typically stimulated byactivation of the T cell receptor (TCR). To determine if DNA-PKcs wasacting in a TCR directed pathway, we repeated the IL-2 production assayafter stimulating T cells with anti-CD28/CD3 dynabeads which activatethe TCR. Cells were harvested 24 hours after stimulation and treatmentwith or without the NU7441 inhibitor. As seen with PMA+PHA activation,NU7441 significantly blocked IL-2 production stimulated by anti-CD28/CD3dynabeads (FIG. 1C). To confirm that the effect of IL-2 secretion wasspecific to DNA-PKcs and not a side effect of the inhibitor, theexpression of DNA-PKcs using short hairpin RNA plasmids (shRNA) wasknocked down. The protein level of DNA-PKcs was reduced with shRNAindicating that the knock down of DNA-PKcs was successful (FIG. 1D).Loss of DNA-PKcs expression significantly inhibited secretion of IL-2 inT cells following activation with PMA+PHA confirming DNA-PKcs as acritical regulator of IL-2 production (FIG. 1D).

Next the effect of DNA-PKcs inhibition on IL-2 production in moreclinically relevant human primary immune cells was examined. Therefore,DNA-PKcs activity in Peripheral Blood Mononuclear Cells, PMBC, wasinhibited and the production of IL-2 was evaluated. Like Jurkat cells,NU7441 did not affect cellular viability but did significantly reducethe level of IL-2 produced following activation with PMA+PHA (FIG. 5 andFIG. 1E).

DNA-PKcs Inhibition Blocks Nuclear Localization of NFAT:

IL-2 production is initiated by dephosphorylation and translocation ofthe transcription factor NFAT to the nucleus. Therefore, the effect ofDNA-PKcs inhibition on NFAT in Jurkat cells by western blot andimmunocytochemistry was examined. In FIG. 2A, it is shown thatactivation of Jurkat cells with PMA+PHA induced phosphorylation ofDNA-PKcs at serine 2056, an activation site [25]. Additionally, NU7441effectively inhibited DNA-PKcs phosphorylation confirming that NU7441successfully inhibits DNA-PKcs activity. Without activation, NFAT wasphosphorylated (s237) and resided in the cytoplasm in Jurkat cells (FIG.2A and FIG. 2B). Upon activation, NFAT was dephosphorylated andtranslocated to the nucleus. However, in the presence of NU7441, NFATremained phosphorylated and nuclear localization was prevented, furthersuggesting that DNA-PKcs is critical for proper T cell signaling (FIG.2A and FIG. 2B).

DNA-PKcs Inhibition Reduces Calcineurin Activity in T Cells:

As mentioned above, the regulation of NFAT is mediated viaphosphorylation. During T cell activation, calcineurin, acalcium/calmodulin-dependent serine-threonine phosphatase, is activatedand dephosphorylates NFAT allowing it to translocate to the nucleus toinitiate transcription. Therefore, the effect of DNA-PKcs on calcineurinactivity in Jurkat cells was evaluated. The phosphatase activity ofcalcineurin was greatly increased in the presence of PMA+PHA, howeverthe activity was significantly inhibited with NU7441 treatment (FIG.3A). Since the activity of calcineurin is regulated by the intracellularCa²⁺ ion concentration, the level of calcium ions was monitored. In thepresence of NU7441 following activation, there was no change in theconcentration of Ca²⁺ ions (FIG. 3B) proving that DNA-PKcs does notregulate calcineurin activity by altering the influx of Ca²⁺ but by adifferent unknown mechanism.

Mammalian target of rapamycin, mTOR, a member of the PI3K kinase family,is a second signaling pathway initiated following T cell activation[26]. Therefore, the effect of DNA-PKcs on the mTOR pathway wasevaluated. Activated Jurkat cells with or without NU7441 treatment weresubjected to an mTOR assay which detects the level of activated mTORwith an antibody specific to phosphorylated ser2448 was evaluated.Results from the assay along with western blot analysis ofphosphorylated mTOR showed that loss of DNA-PKcs activity did not altermTOR activation in T cells. (FIG. 3C). This further indicates a functionfor DNA-PKcs in T cells that is specific to the calcineurin signalingpathway.

DNA-PKcs Regulates Expression of the Calcineurin Inhibitor Cabin 1:

The endogenous calcineurin inhibitor, Cabin1, binds calcineurinpreventing the dephosphorylation of NFAT and transcription of immunecytokines including IL-2 [19, 20]. Cabin1 works in a similar fashion inthe DNA damage repair pathway by binding p53 preventing its interactionwith DNA [21, 22]. In this pathway, Cabin1 expression is controlled bycheckpoint kinase CHK2. DNA damage signals the phosphorylation of CHK2by DNA-PKcs at site T68 which stimulates CHK2 to hyper-phosphorylateCabin1 targeting it for ubiquitination and degradation [23]. In thisstudy, the relationship between DNA-PKcs, CHK2, and Cabin1 in the T cellsignaling pathway was examined. We show that following activation of Tcells, phosphorylation of DNA-PKcs (s2056) is increased along with anincrease in CHK2 phosphorylation at Thr68 (FIG. 4A). Phosphorylation ofboth proteins was reduced with the NU744 inhibitor (FIG. 4A) indicatingthat DNA-PKcs is partly responsible for CHK2 activation in T cells.However, inhibition of DNA-PKcs and subsequently activation of CHK2caused an increase in Cabin1 expression (FIG. 4A). This effect wouldresult in a decrease in calcineurin activity and IL-2 production. Thesedata highlight a novel mechanism by which DNA-PKcs regulates calcineurinsignaling in T cells by its inhibitor Cabin1. A schematic of thismechanism is displayed in FIG. 4B.

Discussion

DNA-PKcs is a ubiquitously expressed enzyme with an increasing amount offunctions defined in the literature. Not only is the enzyme criticallyimportant for NHEJ, but it has also been shown to phosphorylate a widevariety of substrates critical to cell growth, division, and homeostasis[10-14]. Mutations of DNA-PKcs in mammals present clinically with a SCIDphenotype that is indistinguishable from other genetic causes of SCID[27]. Given its emerging function as a key regulator for numeroussignaling transduction pathways, we hypothesized that DNA-PKcs not onlyaffects the immune response through its role in V(D)J recombination butalso by regulation of the calcineurin signaling pathway which stimulatesthe production of IL-2, a critical immune cell cytokine. Interestingly,DNA-PKcs has been previously reported to associate with proteins thatbind to the antigen receptor response element in the IL-2 promoterregion further suggesting a role for this protein in IL-2 regulation[28]. The IL-2 pathway has been extensively researched and hassignificant clinical importance particularly with respect to transplant,cancer, and cardiovascular biology. DNA-PKcs has not previously beenlinked to either mature T cell activation or the calcineurin signalingpathway. Using a Jurkat T cell model, a novel mechanism where DNA-PKcsregulates T cell-mediated signaling by altering the expression of thecalcineurin inhibitor, Cabin1 was identified. The function of Cabin1 inT cell signaling has been well-characterized as a negative regulator ofcalcineurin activity [19, 20]. It was shown that through Cabin1,DNA-PKcs can exert control over the immune response. Like DNA-PKcs andCHK2, Cabin1 is involved in the DNA damage repair pathway. Cabin1functions to inhibit the pathway by binding to p53 preventing itsability to bind DNA and promote transcription of DNA repair genes [21,22]. Expression of Cabin1 is altered in response to DNA damage throughactivation of ATM and its target kinase, CHK2. Phosphorylation andactivation of CHK2 result in degradation of Cabin1 freeing p53 to bindto DNA. DNA-PKcs has not been shown to effect Cabin1 expression,however; it does phosphorylate and activate CHK2 in response to DNAdamage [23].

The results presented here underscore an additional role of DNA-PKcs inthe immune system. Small molecule inhibition of DNA-PKcs is currently inPhase I clinical trials for cancer therapy with the idea being thatchemoresistance can be usurped via disruption of a DNA double strandbreak repair pathway [32] (Clinicaltrials.gov). Without being bound bytheory, the results suggest that inhibition of this enzyme will likelyhave an immediate and profound effect on T-cell signaling in addition toits well-established role in V(D)J recombination. While the outcome ofthese clinical trials and the benefit of DNA-PKcs inhibitors as cancertherapy are still being evaluated, one could hypothesize the outcome.Loss of IL-2 expression due to these inhibitors could result in areduced anti-oncogenic T cell response counteracting any positive effectfrom the inhibition of DNA damage repair. The effect of DNA-PKcs on IL-2production must be considered when deciphering the outcome of thesetrials.

This work also highlights a novel use for DNA-PKcs inhibitors. Singledrug small molecule inhibition of both cell mediated and humoralimmunity is a goal of transplant pharmacology. Given this data, we feelthat DNA-PKcs is a worthwhile target for immunosuppression in thetransplant population as both an induction agent and possiblemaintenance therapy. Results from this study warrant investigation intothe immunosuppression benefit of DNA-PKcs inhibition in transplantrecipients.

REFERENCES

-   1. Burma S, Chen D J. Role of DNA-PK in the cellular response to DNA    double-strand breaks. DNA Repair 2004; 3(8-9):909-18.-   2. Davis A J, So S, Chen D J. Dynamics of the PI3K-like protein    kinase members ATM and DNA-PKcs at DNA double strand breaks. Cell    Cycle 2010; 9(13):2529-36.-   3. Davis A J, Chen D J. DNA double strand break repair via    non-homologous end-joining. Transl Cancer Res 2013; 2(3):130-143.-   4. Taccioli G E, Amatucci A G, Beamish H J, Gell D, Xiang X H,    Torres Arzayus M I, et al. Targeted disruption of the catalytic    subunit of the DNA-PK gene in mice confers severe combined    immunodeficiency and radiosensitivity. Immunity 1998; 9(3):355-66.-   5. Jhappan C, Morse H C, 3rd, Fleischmann R D, Gottesman M M,    Merlino G. DNA-PKcs: a T-cell tumour suppressor encoded at the mouse    scid locus. Nat Genet 1997; 17(4):483-6.-   6. Gao Y, Chaudhuri J, Zhu C, Davidson L, Weaver D T, Alt F W. A    Targeted DNA-PKcs-Null Mutation Reveals DNA-PK-Independent Functions    for KU in V(D)J Recombination. Immunity 1998; 9(3):367-376-   7. Alt F W, Yancopoulos G D, Blackwell T K, Wood C, Thomas E, Boss    M, et al. Ordered rearrangement of immunoglobulin heavy chain    variable region segments. Embo J 1984; 3(6):1209-19.-   8. Born W, Yague J, Palmer E, Kappler J, Marrack P. Rearrangement of    T-cell receptor beta-chain genes during T-cell development. Proc    Natl Acad Sci USA 1985; 82(9):2925-9.-   9. DiSanto J P, Dautry-Varsat A, Certain S, Fischer A, de Saint    Basile G. Interleukin-2 (IL-2) receptor gamma chain mutations in    X-linked severe combined immunodeficiency disease result in the loss    of high-affinity IL-2 receptor binding. Eur J Immunol 1994;    24(2):475-9.-   10. Minjgee M, Toulany M, Kehlbach R, Giehl K, Rodemann H P.    K-RAS(V12) induces autocrine production of EGFR ligands and mediates    radioresistance through EGFR-dependent Akt signaling and activation    of DNA-PKcs. Int J Radiat Oncol Biol Phys 2011; 81(5):1506-14.-   11. Burdine L J, Burdine M S, Moreland L, Fogel B, Orr L M, James J,    et al. Proteomic Identification of DNA-PK Involvement within the RET    Signaling Pathway. PLoS One 2015; 10(6).-   12. Toulany M, Lee K J, Fattah K R, Lin Y F, Fehrenbacher B,    Schaller M, et al. Akt promotes post-irradiation survival of human    tumor cells through initiation, progression, and termination of    DNA-PKcs-dependent DNA double-strand break repair. Mol Cancer Res    2012; 10(7):945-57.-   13. Dragoi A M, Fu X, Ivanov S, Zhang P, Sheng L, Wu D, et al.    DNA-PKcs, but not TLR9, is required for activation of Akt by    CpG-DNA. Embo J 2005; 24(4):779-89.-   14. Wong R H, Chang I, Hudak C S, Hyun S, Kwan H Y, Sul H S. A role    of DNA-PK for the metabolic gene regulation in response to insulin.    Cell 2009; 136(6):1056-72.-   15. Boyman O, Sprent J. The role of interleukin-2 during homeostasis    and activation of the immune system. Nat Rev Immunol 2012;    12(3):180-90.-   16. Hogan P G, Chen L, Nardone J, Rao A. Transcriptional regulation    by calcium, calcineurin, and NFAT. Genes Dev 2003; 17(18):2205-32.-   17. Liao W, Lin J X, Leonard W J. IL-2 family cytokines: new    insights into the complex roles of IL-2 as a broad regulator of T    helper cell differentiation. Curr Opin Immunol 2011; 23(5):598-604.-   18. Malek T R, Castro I. Interleukin-2 receptor signaling: at the    interface between tolerance and immunity. Immunity 2010;    33(2):153-65.-   19. Sun L, Youn H D, Loh C, Stolow M, He W, Liu J O. Cabin 1, a    negative regulator for calcineurin signaling in T lymphocytes.    Immunity 1998; 8(6):703-11.-   20. Esau C, Boes M, Youn H D, Tatterson L, Liu J O, Chen J. Deletion    of calcineurin and myocyte enhancer factor 2 (MEF2) binding domain    of Cabin1 results in enhanced cytokine gene expression in T cells. J    Exp Med 2001; 194(10):1449-59.-   21. Jang H, Choi S Y, Cho E J, Youn H D. Cabin1 restrains p53    activity on chromatin. Nat Struct Mol Biol 2009; 16(9):910-5.-   22. Choi S Y, Jang H, Roe J S, Kim S T, Cho E J, Youn H D.    Phosphorylation and ubiquitination-dependent degradation of CABIN1    releases p53 for transactivation upon genotoxic stress. Nucleic    Acids Res 2013; 41(4):2180-90.-   23. Li J, Stern D F. Regulation of CHK2 by DNA-dependent protein    kinase. J Biol Chem 2005; 280(12):12041-50.-   24. Leahy J J, Golding B T, Griffin R J, Hardcastle I R, Richardson    C, Rigoreau L, et al. Identification of a highly potent and    selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441)    by screening of chromenone libraries. Bioorganic and Medicinal    Chemistry Letters 2004; 14(24):6083-7.-   25. Chen B P, Chan D W, Kobayashi J, Burma S, Asaithamby A,    Morotomi-Yano K, et al. Cell cycle dependence of DNA-dependent    protein kinase phosphorylation in response to DNA double strand    breaks. Journal of Biological Chemistry 2005; 280(15):14709-15.-   26. Dennis P B, Fumagalli S, Thomas G. Target of rapamycin (TOR):    balancing the opposing forces of protein synthesis and degradation.    Current Opinion in Genetics and Development 1999; 9(1):49-54.-   27. van der Burg M, van Dongen J J, van Gent D C. DNA-PKcs    deficiency in human: long predicted, finally found. Curr Opin    Allergy Clin Immunol 2009; 9(6):503-9.-   28. Ting N S, Kao P N, Chan D W, Lintott L G, Lees-Miller S P.    DNA-dependent protein kinase interacts with antigen receptor    response element binding proteins NF90 and NF45. Journal of    Biological Chemistry 1998; 273: 2136-2145.-   29. Hein A L, Ouellette M M, Yan Y. Radiation-induced signaling    pathways that promote cancer cell survival (review). International    Journal of Oncology 2014; 45(5):1813-9.-   30. Golding S E, Morgan R N, Adams B R, Hawkins A J, Povirk L F,    Valerie K. Pro-survival AKT and ERK signaling from EGFR and mutant    EGFRvIII enhances DNA double-strand break repair in human glioma    cells. Cancer Biol Ther 2009; 8(8):730-8.-   31. Chen B P, Uematsu N, Kobayashi J, Lerenthal Y, Krempler A,    Yajima H, et al. Ataxia telangiectasia mutated (ATM) is essential    for DNA-PKcs phosphorylations at the Thr-2609 cluster upon DNA    double strand break. Journal of Biological Chemistry 2007;    282(9):6582-7.-   32. Van Triest B, Damstrup L, Falkenius J, Budach V, Troost E,    Samuels M, et al. A phase 1a/1b trial of the DNA-dependent protein    kinase inhibitor (DNA-PKi) M3814 in combination with radiotherapy in    patients with advanced solid tumors. Journal of Clinical Oncology    2017; 35: e14048-e14048.

Example 2: Inhibition of DNA-PKcs as Immunosuppression Therapy forTransplant Patients Methods and Materials

Materials:

DNA-PK inhibitor, NU7441 was purchased from Selleckchem.com and used invivo at 10 mg/kg diluted in DMSO and 40% PEG or in vitro at 5 μM.Tacrolimus (FK506) was ordered for Sigma Aldrich and used at aconcentration of 1 mg/kg in vivo.

Mouse Skin Allograft:

To confirm that DNA-PK inhibitors could function as an immunosuppressantin vivo, mouse skin allograft studies were performed. All studies wereperformed under an approved IACUC protocol. Ears from donor BALBc micewere removed and split into two removing all cartilaginous material. Theear skin was placed on an incision made to the back of a recipientC571316 or DNA-PK knockout (NOD.CB17-Prkdc scid/J-20) mouse. The skinwas sutured in 4 places using 6-0 nylon sutures. Wounds were wrapped ingauze and ban-daids and allowed to heal for 6 days before removing thebandage. Spleens were harvested from donor mice for splenocyteisolation. On Day 0 through Day 10 (the day of sacrifice) mice wereinjected (100 ul) daily with one of the following: saline, Tacrolimus (1mg/kg), or NU7441 (1 mg/kg). Mice were monitored for rejection dailywhich was indicated by dark, black appearance. At the time of sacrifice,mice were euthanisized according to IACUC guidelines and skin graftsremoved and fixed in formalin for histology. Spleens and blood were alsocollected.

Evaluation of PD-1 and IL-2 Expression:

The effect of NU7441 on the expression of PD-1, a protein withimmunosuppressant function, and IL-2 was examined in Jurkat cellsfollowing stimulation with PHA+PMA and treatment with the inhibitorNU7441. qPCR analysis was performed using primers specific for PD1 andIL-2 sequences.

qPCR on T Cell Markers:

Naïve CD4+ T cells were isolated by standard methods from the spleens ofBalbc mice. T cells were grown in culture and treated with cytokinesspecific for differentiate into Th1 and Th17 subtypes. Additionally, Tcells were cultured with NU7441 and FK506. qPCR was performed looking atthe following markers: Batf3, RORyt, IL17, IL22, TGFBeta, Lymphotoxin,T-Bet.

Results

In the provided data we show that treatment with NU7441, a DNA-PKinhibitor, reduces rejection in a skin allograft mouse model compared tocontrol groups treated only with saline. Visual examination of salinegroups confirmed severe necrosis while NU7441 treatment groups had amajority with 0% necrosis (FIG. 6A). On Day 10 at the completion of thestudy, DNA-OK KO mice and NU7441 treated mice had a significantreduction in the level of necrosis with controls being around 80% onaverage and NU7441 around 30% and KO around 15-20% (FIG. 6B and FIG.6C). This data confirms that DNA-PK inhibition is a legitimate means forimmunosuppression following transplantation. Additionally, to understandbetter how DNA-PK is regulating the immune system, studies wereperformed to analyze the effect of DNA-PK inhibition on PD-1 expressiongiven that PD-1 is a protein with immunosuppressant functions. Treatmentwith NU7441 did not alter the expression level of PD-1 althoughtreatment with the calcineurin inhibitor FK506 did significantly reduceexpression. IL-2 expression was reduced with both NU7441 and FK506 asexpected given our prior studies showing NU7441 reduces 11-2 expressionby inhibiting calcineurin activity (FIG. 7A and FIG. 7B). Next, studieswere performed to analyze the effects of DNA-PK inhibition on T cellsubtype differentiation since T cell subtypes often regulate immunityand disease outcome. qPCR analysis of specific subtype markers followingtreatment with different cytokines for differentiation along with NU7441or FK506 indicated that NU7441 was promoting the expression of Th1 Tcells while suppressing a Th17 environment. (FIG. 8A, FIG. 8B, FIG. 8C,FIG. 8D, and FIG. 9E). This information provides evidence that DNA-PK isa key regulator of the immune response and can alter the population of Tcell subtype.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments have been described above for purposes ofillustration, it will be appreciated by those skilled in the art thatnumerous variations of the details may be made without departing fromthe disclosure as described in the appended claims.

1. A method of improving the transplantation outcome in a subjectreceiving an organ transplant, the method comprising administering tothe subject a therapeutically effective amount of a compositioncomprising a DNA-PK inhibitor, wherein the DNA-PK inhibitor improves thetransplantation outcome.
 2. The method of claim 1, wherein theimprovement in transplantation outcome is reduced graft rejection orincreased graft survival.
 3. The method of claim 2, wherein the reducedgraft rejection is reduced chronic graft rejection or reduced acutegraft rejection.
 4. (canceled)
 5. The method of claim 1, wherein theorgan transplant is an allograft or xenograft. 6.-7. (canceled)
 8. Themethod of claim 1, wherein the DNA-PK inhibitor suppresses T or B cellactivity.
 9. The method of claim 8, wherein the DNA-PK inhibitorsuppresses T and B cell activity. 10.-11. (canceled)
 12. The method ofclaim 1, wherein the DNA-PK inhibitor suppresses IL-2 secretion inT-cells.
 13. (canceled)
 14. The method of claim 1, wherein theimprovement in transplantation outcome is decreased lymphocyticinfiltration, vasculitis, infarction, ischemia, thrombosis, intimalthickening, glomerular atrophy, glomerular sclerosis, tubular atrophy,hyalinization, interstitial fibrosis, cortical fibrosis, serumcreatinine levels, intimal proliferation, hypertrophy, cardiac vesseldisease post-transplant, graft intimal hyperplasia, luminal occlusion,or bronchitis obliterans.
 15. The method of claim 1, wherein the subjectis the recipient of an organ tissue transplant, said organ tissueselected from the group consisting of kidney, liver, heart, lung, bonemarrow, and cornea tissue.
 16. The method of claim 1, wherein the DNA-PKinhibitor composition is administered by intravascular, intravenous,intra-arterial, subcutaneous, intramuscular, intraperitoneal,intraventricular, or intraepidural administration.
 17. (canceled) 18.The method of claim 1, wherein the DNA-PK inhibitor is selected from thegroup consisting of a small-molecule inhibitor, nucleotide inhibitor,and antibody inhibitor, wherein the DNA-PK inhibitor reduces DNA-PKactivity.
 19. A method of reducing immune response in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of a composition comprising a DNA-PKinhibitor, wherein the DNA-PK inhibitor reduces an immune response. 20.The method of claim 19, wherein the DNA-PK inhibitor reduces or blocksan immune response already in progress.
 21. The method of claim 19,wherein the DNA-PK inhibitor prevents the induction of an immuneresponse.
 22. The method of claim 19, wherein the DNA-PK inhibitorsuppresses T or B cell activity.
 23. The method of claim 19, wherein theDNA-PK inhibitor suppresses T and B cell activity. 24.-25. (canceled)26. The method of claim 19, wherein the DNA-PK inhibitor is selectedfrom the group consisting of a small-molecule inhibitor, nucleotideinhibitor, and antibody inhibitor, wherein the DNA-PK inhibitor reducesDNA-PK activity.
 27. The method of claim 19, wherein the DNA-PKinhibitor composition is administered orally, parenterally, ortopically.
 28. (canceled)
 29. A method of treating an IL-2 relateddisease in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of acomposition comprising a DNA-PK inhibitor, wherein the DNA-PK inhibitorsuppresses humoral or cellular immunity.
 30. The method of claim 29,wherein the IL-2 related disease is one or more of AIDS, psoriasis,atopic dermatitis, urticaria, lupus nephritis, allergic conjunctivitis,sty, chalazion, spring catarrh, uveitis, polymyositis, Hashimoto'sdisease, Behcet's disease, ankylosing spondylitis, systemic sclerosis,Sjogren's syndrome, pollenosis, scleroderma), gastrointestinal diseases,gout, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis,asthma, chronic obstructive pulmonary disease, fibromyalgia, myastheniagravis, sarcoidosis, nasal inflammation and nasal catarrh. 31.-32.(canceled)